The microtubule cytoskeleton: A validated target for the development of 2-Aryl-1H-benzo[d]imidazole derivatives as potential anticancer agents.

In this study, a series of 2-Aryl-1H-benzo[d]imidazole derivatives were developed to target intra- and extracellular microtubule networks. Compounds O-7 and O-10 showed impressive anti-proliferative activity across various tested cell lines, demonstrating selectivity indexes of 151.7 and 61.9, respectively. O-7 achieved an IC50 value of 0.236 ± 0.096 µM, while O-10 showed an IC50 value of 0.622 ± 0.13 µM against A549 cell lines. The induction of early-stage apoptosis in a dose-dependent manner further underscored the potential of O-7 and O-10 as effective anti-proliferative agents. O-7 and O-10 exhibited substantial inhibition of wound closure, with wound closure percentages decreasing from 23% at 0 µM to 0.43% and 2.62% at 20 µM, respectively. Colony formation reduction rates were impressive, with O-7 at 74.2% and O-10 at 81.2%. These results indicate that the O-7 and O-10 can impede cancer cell migration and have a high potential to curtail colony formation. The mode of action investigations for O-7 and O-10 revealed that O-7 could inhibit in vitro tubulin polymerization and disrupt the intracellular microtubule cytoskeleton. This disruption led to cell cycle arrest in the G2/M phase, indicating that O-7 exerts its anticancer activity through microtubule destabilization. However, O-10 shows a different mode of action than O-7 and requires further investigation. Overall, our study showcases the potential of the synthesized benzimidazole derivatives as novel and selective anticancer agents, motivating further exploration of their pharmacological properties and therapeutic applications.

An indole-based near-infrared fluorescent "Turn-On" probe for H2O2: Selective detection and ultrasensitive imaging of zebrafish gallbladder.

Fluorescent probes play essential roles in medical imaging, where the researchers can select one of many molecules to use to help monitor the status of living systems under investigation. To date, a few scaffolds that allow the in vivo detection of H2O2 are available only. Herein, we provide a highly sensitive and selective near-infrared fluorescent probe that detects H2O2 based on the ICT sensing mechanism. We report the first indole-incorporated fluorescent probe Indo-H2O2 that allows H2O2 detection with a LOD of 25.2 nM featuring a boronate group conjugated to an indole scaffold; the boronate cleaves upon reaction with H2O2. A 5-membered malononitrile derivative was incorporated; Indo-H2O2 has near-infrared (NIR) properties and the reaction time is low (∼25 min) compared to other related probes. Indo-H2O2 was successfully employed in both endogenous and exogenous imaging trials of H2O2 in living cells. Indo-H2O2 also allows the real-time monitoring of H2O2in vivo. It preferentially accesses the gallbladder of zebrafish. Our findings support Indo-H2O2 as a highly sensitive fluorescent NIR probe for detecting H2O2, and an idea to incorporate a central indole unit in future fluorescent probe designs.

Highly sensitive and rapid detection of hypochlorous acid in aqueous media and its bioimaging in live cells and zebrafish using an ESIPT-driven mycophenolic acid-based fluorescent probe.

Excessive production of potent biological oxidants such as HOCl has been implicated in numerous diseases. Thus, it is crucial to develop highly specific and precise methods to detect HOCl in living systems, preferably with molecules that can show a distinct therapeutic effect. Our study introduces the synthesis and application of a highly sensitive fluorescence "turn-on" probe, Myco-OCl, based on the mycophenolic acid scaffold with exceptional water solubility. The ESIPT-driven mechanism enables Myco-OCl to specifically and rapidly detect (<5 s) HOCl with an impressive Stokes shift of 105 nm (λex = 417 nm, λem = 522 nm) and a sub-nanomolar (97.3 nM) detection limit with the detection range of 0 to 50 µM. The potential of Myco-OCl as an excellent biosensor is evident from its successful application for live cell imaging of exogenous and endogenous HOCl. In addition, Myco-OCl enabled us to detect HOCl in a zebrafish inflammatory animal model. These underscore the great potential of Myco-OCl for detecting HOCl in diverse physiological systems. Our findings thus offer a highly promising tool for detecting HOCl in living organisms.

A dual-function fluorescence 'turn-on' probe that allows Zn (II) bioimaging and quantification of water in the organic solvent.

Herein, a Eugenol-derived fluorescence 'turn-on' probe FLHE was synthesized by condensing 2-((3-(trifluoromethyl)phenyl)amino)benzohydrazide with 5-allyl-2-hydroxy-3-methoxybenzaldehyde. FLHE demonstrated very low fluorescence in the studied organic solvents of varying polarities. However, upon titration with Zn2+ in HEPES buffer (pH = 7.4, 50% ACN, v/v), FLHE showed 40-fold higher fluorescence signals indicating the formation of the FLHE-Zn2+ complex. The fluorescence turn-on phenomenon upon FLHE-Zn2+ complex formation results from a chelation-enhanced fluorescence (CHEF) effect. The FLHE-Zn2+ complexation demonstrated a stokes shift of 156 nm (λex = 350 nm, λem = 506 nm) and an about 33-fold increase in the quantum yield (FLHE, Φ = 0.007; FLHE-Zn2+ complex, Φ = 0.23). The binding constant (Ka) determined by the Benesi-Hildebrand plot for interaction between FLHE and Zn2+ was 5.33 × 103 M-1. FLHE demonstrated a LOD of 31.8 nM for detecting Zn2+ in the environmental samples without interference from other cations and anions. FLHE-based paper strip (FLHE-PS) assay was developed to quantify the Zn2+ ions in water and the water content of organic solvent. FLHE-PS allows the detection of Zn2+ in aqueous solutions with a LOD of 63.2 nM and quantifying water in acetonitrile with a LOD of 0.14%. These results indicate that the FLHE has high applicability for detecting Zn2+ in living cells and environmental samples and detecting the presence of water in the organic solvents.

Not all benzimidazole derivatives are microtubule destabilizing agents.

In recent years, microtubule-targeting agents (MTAs) have gained considerable interest in developing novel small-molecule anticancer drugs. MTAs demonstrate anticancer activity either as microtubule-stabilizing agents (paclitaxel) or microtubule-destabilizing agents (nocodazole). FDA-approved drugs containing a benzimidazole ring (nocodazole, albendazole, mebendazole, etc.) are well-known microtubule-destabilizing agents. Thus, most recent research on benzimidazole scaffold-based MTAs focuses on developing microtubule-destabilizing agents. However, there is no report on the benzimidazole scaffold-based microtubule-stabilizing agent. Here, we present the benzimidazole derivatives NI-11 and NI-18 that showed a profound anticancer activity as microtubule-stabilization agents. About twenty benzimidazole analogues were synthesized with excellent yield (80.0% ∼ 98.0%) and tested for their anticancer activity using two cancer cell lines (A549, MCF-7) and one normal cell line (MRC-5). NI-11 showed IC50 values of 2.90, 7.17, and 16.9 µM in A549, MCF-7, and MRC-5 cell lines. NI-18 showed IC50 values of 2.33, 6.10, and 12.1 µM in A549, MCF-7, and MRC-5 cell lines. Thus, NI-11 and NI-18 demonstrated selectivity indexes of 5.81 and 5.20, respectively, which are much higher than the currently available anticancer agents. NI-11 and NI-18 inhibited the cancer cell motility and migration, induced the early phase apoptosis. Both of these comounds were found to show an upregulation of DeY-α-tubulin and downregulation of Ac-α-tubulin expressions in cancer cells. Eventhough the reported benzimidazole scaffold-based commercially available drugs are known to be microtubule-destabilizing agents, the analogues NI-11 and NI-18 were found to have microtubule-stabilizing activity. The in vitro tubulin polymerization assay and the immunofluorescence assay results indicate that the NI-11 and NI-18 exhibit anticancer activity by stabilizing the microtubule network.

Exploration of Molecular Structure, DFT Calculations, and Antioxidant Activity of a Hydrazone Derivative.

The hydrazine derivatives are known to possess several biological activities including anticancer, antibacterial and anti-fungal, anticonvulsant, and antioxidant. This communication presents the synthesis, X-ray crystal structure analysis, DFT calculations, cell cytotoxicity, and antioxidant activity of the Schiff base 4,4'-((1E,1'E)-hydrazine-1,2-diylidenebis(ethan-1-yl-1-ylidene))bis(benzene-1,3-diol) (compound 2). We have also isolated the side product compound 1 and characterized it using single X-ray crystallography. The crystal structure of compound 1 depicts that the ensuing C-H···N hydrogen bonding interaction is presented and discussed herein. In addition, the calculations using density functional theory (DFT) approximation supported by experimental 1H and 13C NMR studies on the key compound 2 are reported. The results of theoretical and experimental 1H and 13C NMR were concordant. The antioxidant activity of compound 2 was determined by using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) radical cation assays and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay. Compound 2 demonstrated excellent antioxidant activity in ABTS assay (IC50 = 4.30 ± 0.21 µM) and DPPH assay (IC50 = 81.06 ± 0.72 µM) with almost no cytotoxicity below 25 µM.

ID-Checker Technology for the Highly Selective Macroscale Delivery of Anticancer Agents to the Cancer Cells.

Cancer cells deploy several glucose transport protein (GLUT) channels on the cell membranes to increase glucose uptake. Cancer cells die within 24 h in the absence of glucose. Thus, preventing the deployment of GLUT channels can deprive them of glucose, resulting in apoptosis within 24 h. Herein, we developed the ID-Checker with a glucose tag that ensures its highly specific macroscale delivery of anticancer agents to the cancer cells through the GLUT channels. ID-Checker presented here showed IC50 values of 0.17-0.27 and 3.34 µM in cancer and normal cell lines, respectively. ID-Checker showed a selectivity index of 12.5-20.2, which is about 10-20 times higher than that of known anticancer agents such as colchicine. ID-Checker inhibits the microtubule formation, which results in the prevention of the deployment of GLUT channels in 6 h and kills the cancer cells within 24 h without any damage to normal cells.

Combination Therapy of Ledipasvir and Itraconazole in the Treatment of COVID-19 Patients Coinfected with Black Fungus: An In Silico Statement.

The manuscript mainly aimed at providing clues on improving the innate immunity of coronavirus patients and safeguarding them from both new mutant strains and black fungus infections. Coronavirus is readily mutating from one variant to another. Among the several variants, we selected SARS-CoV-2 B.1.1.7 in this study. Upon infection of any virus, ideally, the phagocytic cells of the host engulf and destroy the virus by a mechanism called phagocytosis. However, compromised immunity impairs phagocytosis, and thus, restoring the immune system is crucial for a speedy recovery of infected patients. The autophagy and activation of Toll-like receptor-4 are the only ways to restore innate immunity. Recently, immunocompromised COVID-19 patients have been suffering from the coinfection of black fungus. Rhizomucor, a black fungus species, causes more than 75% of cases of mucormycosis. Here, we present the results of molecular docking studies of sixty approved antiviral drugs targeting receptors associated with the SARS-CoV-2 B 1.1.7 variant (PDB id: 7NEH), activating the innate immune system (PDB id: 5YEC and 5IJC). We also studied the twenty approved antifungal drugs with Rhizomucor miehei lipase propeptide (PDB id: 6QPR) to identify the possible combination therapy for patients coinfected with coronavirus and black fungus. The ledipasvir showed excellent docking interactions with the 7NEH, 5YEC, and 5IJC, indicating that it is a perfect candidate for the treatment of COVID-19 patients. Itraconazole showed significant interaction with 6QPR of Rhizomucor miehei, suggesting that itraconazole can treat black fungus infections. In conclusion, the combination therapy of ledipasvir and itraconazole can be a better alternative for treating COVID-19 patients coinfected with black fungus.

Elimination Reaction-Based Benzimidazole Probe for Cysteine Detection and Its Application in Serum Sample Analysis.

Benzimidazole-based compound 2-(p-tolyl)-1H-benzo[d]imidazole (3) and its derivative probe A-B have been synthesized for the highly selective detection and quantification of Cys in human serum. The photophysical properties of A-B and compound 3 were evaluated by UV-vis absorption and fluorescence spectroscopy. A-B showed high selectivity and sensitivity for Cys among tested analytes, including amino acids, anions, and cations. A-B selectively reacts with Cys and results in compound 3 with fluorescence turn-on effect. A-B did not show any interference from the components in the serum matrix for Cys detection in the human serum sample. A-B detects Cys in serum samples with 2.3-5.4-fold better LOD than reported methods. The detection limit of 86 nM and 43 nM in HEPES buffer using UV-visible and fluorescence spectroscopy, respectively, makes A-B an excellent chemosensor for Cys detection.

Detection and Quantification of Tp53 and p53-Anti-p53 Autoantibody Immune Complex: Promising Biomarkers in Early Stage Lung Cancer Diagnosis.

Lung cancer is a leading cause of death worldwide, claiming nearly 1.80 million lives in 2020. Screening with low-dose computed tomography (LDCT) reduces lung cancer mortality by about 20% compared to standard chest X-rays among current or heavy smokers. However, several reports indicate that LDCT has a high false-positive rate. In this regard, methods based on biomarker detection offer excellent potential for developing noninvasive cancer diagnostic tests to complement LDCT for detecting stage 0∼IV lung cancers. Herein, we have developed a method for detecting and quantifying a p53-anti-p53 autoantibody complex and the total p53 antigen (wild and mutant). The LOD for detecting Tp53 and PIC were 7.41 pg/mL and 5.74 pg/mL, respectively. The detection ranges for both biomarkers were 0-7500 pg/mL. The known interfering agents in immunoassays such as biotin, bilirubin, intra-lipid, and hemoglobin did not detect Tp53 and PIC, even at levels that were several folds higher levels than their normal levels. Furthermore, the present study provides a unique report on this preliminary investigation using the PIC/Tp53 ratio to detect stage I-IV lung cancers. The presented method detects lung cancers with 81.6% sensitivity and 93.3% specificity. These results indicate that the presented method has high applicability for the identification of lung cancer patients from the healthy population.

Synthesis and evaluation of 2-aryl-1H-benzo[d]imidazole derivatives as potential microtubule targeting agents.

Microtubule targeting agents (MTAs) are the potential drug candidates for anticancer drug discovery. Disrupting the microtubule formation or inhibiting the de-polymerization process by a synthetic molecule can lead to an excellent anticancer drug candidate. Here, we present the 2,5-substituted-1H-benzo[d]imidazole derivatives as potential colchicine, nocodazole binding site targeting agents. About 20 benzimidazole derivatives were synthesized with 82.0%-94.0% yield using mild reaction conditions. The synthesized compounds showed moderate to excellent anticancer activity established in three cell lines, including Hela cells, A549 cells, MRC-5 cells. The compounds B15, B16, B19, and B20 are the potential candidates with the IC50 values <15 µM in the three different cell lines. In MTT assay, compounds B15, B16, B19, and B20 showed excellent antiproliferation activity indicated by IC50 values in the range of 5.3 ± 0.21 to 18.1 ± 0.32 µM using HeLa and A549 cell lines. The predicted absorption, distribution, metabolism and excretion (ADME) properties and drug-likeness properties of B15, B16, B19, and B20 indicate that these compounds can be used as lead compounds for further study to develop excellent MTAs.

Indazole Derivatives Effective against Gastrointestinal Diseases.

BACKGROUND: In this fast-growing lifestyle, humans are in the race against time to cope up with busy schedule. Less exercise, consumption of high calorie-low fiber food and stress take us one step closer towards digestive dysfunction. Dysfunctional digestive system causes various gastrointestinal disorders like constipation, IBS, UC, diarrhea, gastrointestinal tract immobility, hyperglycemia, hemorrhoids, fistula, anal fissures, stomach cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer and metabolic syndrome. Amongst various natural and synthetic indazole derivatives nigellicine, nigellamine, nigellidine, zanubrutinib and SCH772984 showed prominent results to cure various gastrointestinal disorders. OBJECTIVES: In this manuscript, we focus on the importance of indazole derivatives in the treatment of various gastrointestinal diseases. RESULTS AND CONCLUSION: In the treatment of IBS, four positions (R1, R2, R3 and R4) of indazole were mainly substituted with aromatic aldehyde/substituted methyl, aromatic acid/formamide, benzamide/ sulfonamide and methyl groups, respectively. In case of diarrhea and metabolic syndrome treatment, substitutions with benzyl/isopropyl/acetaldehyde (R1 position) and carboxamide/ formamide (R2 position) of indazole play a critical role. Also, in the treatment of diabetes melitus, all six positions of indazole derivative were substituted with substituted aryl/alkyl/aromatic acid, substituted formamide, substituted acetamide/hydrazide group, halo aryl, substituted aryl/aromatic acid and a long chain of alkyl-aryl alcohol groups, respectively. In the treatment of gastrointestinal cancers, all six positions of indazole derivative were substituted with benzylamide (R1), octanediamide/ benzamide/formamide (R2), carbaldehyde (R4) and substituted phenyl (R5 and R6) groups, respectively. Six receptors (6NP0, 2YME, 4EFU, 4WZ8, 5U4W and 7KKP) associated with GI disorders (co-crystallized with indazole derivative) were identified. Analysis of the receptors showed that co-crystalized ligand molecules were well-interacted with receptors via pie-pie interaction, coordinate and sigma bonding within 4 Å distance. As per Ramachandran plot analysis, more than 90% of the amino acid residues were present in the most favored region. So, if sufficient focuses are imposed on the development of newer indazole derivatives to treat gastrointestinal diseases, it will work as a boon to society.

An abiotic fluorescent probe for the detection and quantification of carcinoembryonic antigen.

The reported methods mainly use biomolecules such as antibodies, enzymes, and aptamers for biomarker detection. However, applying an abiotic fluorescent probe to detect cancer biomarkers such as carcinoembryonic antigen (CEA) has not been reported. In this regard, we conceived an abiotic fluorescent probe BIQ-1 for the rapid yet straightforward detection of CEA. The bioinformatics tools and molecular docking techniques were used to develop the probe BIQ-1 for the selective detection and quantification of CEA in a buffer matrix resembling serum. The probe BIQ-1 exhibited a limit of detection of 0.2 ng/mL for CEA in a simple cuvette-based experiment. The BIQ-1 did no show interference from the possible interfering components such as hemoglobin, intralipid, and human serum albumin (HSA) in concentrations several-fold higher (µg/mL) than CEA.

Development of a Novel Benzimidazole-Based Probe and Portable Fluorimeter for the Detection of Cysteine in Human Urine.

The measurement of cysteine in human urine and live cells is crucial for evaluating biological metabolism, monitoring and maintaining the immune system, preventing tissue/DNA damage caused by free radicals, preventing autoimmune diseases, and diagnosing disorders such as cystinuria and cancer. A method that uses a fluorescence turn-on probe and a portable fluorescence spectrometer device are crucial for highly sensitive, simple, rapid, and inexpensive cysteine detection. Herein, we present the synthesis and application of a benzimidazole-based fluorescent probe (ABIA) along with the design and development of a portable fluorescence spectrometer device (CysDDev) for detecting cysteine in simulated human urine. ABIA showed excellent selectivity and sensitivity in detecting cysteine over homocysteine, glutathione, and other amino acids with the response time of 1 min and demonstrated a detection limit of 16.3 nM using the developed CysDDev. Further, ABIA also demonstrated its utility in detecting intracellular cysteine, making it an excellent probe for bio-imaging assay.

A Novel Method That Allows SNP Discrimination with 160:1 Ratio for Biosensors Based on DNA-DNA Hybridization.

Highly sensitive (high SBR) and highly specific (high SNP discrimination ratio) DNA hybridization is essential for a biosensor with clinical application. Herein, we propose a method that allows detecting multiple pathogens on a single platform with the SNP discrimination ratios over 160:1 in the dynamic range of 101 to 104 copies per test. The newly developed SWAT method allows achieving highly sensitive and highly specific DNA hybridizations. The detection and discrimination of the MTB and NTM strain in the clinical samples with the SBR and SNP discrimination ratios higher than 160:1 indicate the high clinical applicability of the SWAT.

9G TestTM Cancer/Lung: A Desirable Companion to LDCT for Lung Cancer Screening.

A complimentary biomarker test that can be used in combination with LDCT for lung cancer screening is highly desirable to improve the diagnostic capacity of LDCT and reduce the false-positive rates. Most importantly, the stage I lung cancer detection rate can be dramatically increased by the simultaneous use of a biomarker test with LDCT. The present study was conducted to evaluate 9G testTM Cancer/Lung's sensitivity and specificity in detecting Stage 0~IV lung cancer. The obtained results indicate that the 9G testTM Cancer/Lung can detect lung cancer with overall sensitivity and specificity of 75.0% (69.1~80.3) and 97.3% (95.0~98.8), respectively. The detection of stage I, stage II, stage III, and stage IV cancers with sensitivities of 77.5%, 78.1%, 67.4%, and 33.3%, respectively, at the specificity of 97.3% have never been reported before. The receiver operating characteristic curve analysis allowed us to determine the population-weighted AUC of 0.93 (95% CI, 0.91-0.95). These results indicate that the 9G testTM Cancer/Lung can be used in conjunction with LDCT to screen lung cancer. Furthermore, obtained results indicate that the use of 9G testTM Cancer/Lung with LDCT for lung cancer screening can increase stage I cancer detection, which is crucial to improve the currently low 5-year survival rates.

Macrocycles and Supramolecules as Antioxidants: Excellent Scaffolds for Development of Potential Therapeutic Agents.

Oxidative stress due to the high levels of reactive oxygen species (ROS) that damage biomolecules (lipids, proteins, DNA) results in acute inflammation. However, without proper intervention, acute inflammation progresses to chronic inflammation and then to several chronic diseases, including cancer, myocardial infarction, cardiovascular diseases, chronic inflammation, atherosclerosis, and more. There has been extensive research on the antioxidants of natural origin. However, there are myriad possibilities for the development of synthetic antioxidants for pharmacological applications. There is an increasing interest in the identification of novel synthetic antioxidants for the modulation of biochemical processes related to ROS. In this regard, derivatives of supramolecules, such as calix[n]arene, resorcinarene, calixtyrosol, calixpyrrole, cucurbit[n]uril, porphyrin etc. are gaining attention for their abilities to scavenge the free radicals. Supramolecular chemistry offers excellent scaffolds for the development of novel antioxidants that can be used to modulate free radical reactions and to improve the disorders related to oxidative stress. This review focuses on the interdisciplinary approach for the design and development of novel synthetic antioxidants based on supramolecular scaffolds, with potentially protective effects against oxidative stress.

Development of a Lateral Flow Strip Membrane Assay for Rapid and Sensitive Detection of the SARS-CoV-2.

The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has threatened public health worldwide. The easy human-to-human transmission of this virus has rapidly evolved into a global pandemic. Therefore, to control the community spread of the virus, it is crucial to identify the infected individuals, including asymptomatic people. Hence, a specific and rapid assay is crucial for the early diagnosis and active monitoring of individuals potentially exposed to SARS-CoV-2 for controlling the COVID-19 outbreak. In this study, we have developed the novel lateral flow strip membrane (LFSM) assay that allows the simultaneous detection of RdRp, ORF3a, and N genes using the PCR product obtained by using the single-tube reverse transcription polymerase chain reaction (RT-PCR). The LFSM assay allows detection of SARS-CoV-2 in 30 min at 25 °C after the RT-PCR with the detection limit of 10 copies/test for each gene. The clinical performance of the LFSM assay for the detection of SARS-Cov-2 was evaluated using 162 clinical samples previously detected by using the commercial assay. The percent positive agreement, percent negative agreement, and overall percent agreement of the LFSM assay with the commercial assay were 100% (94.2-100%), 99.0% (94.6-100%), and 99.4% (96.6-100%), respectively. Therefore, the results of the LFSM assay showed significantly high concordance with the commercial assay for the detection of SARS-CoV-2 in clinical specimens. Therefore, we conclude that the developed LFSM assay can be used alone or complementary to the RT-PCR or other methods for the diagnosis and monitoring of the patients to curb community transmission and the pandemic.

Natural Melanin Produced by the Endophytic Bacillus subtilis 4NP-BL Associated with the Halophyte Salicornia brachiata.

Natural melanin with many interesting properties has potential applications in cosmetics, drug delivery, semiconductors, etc. However, conventional production methods are not efficient, resulting in its high cost (350-650 USD g-1), which has been a bottleneck for its efficient commercial utilization. To explore a faster extraction method with a higher yield, a melanin-producing endophytic bacterium was isolated from the halophyte Salicornia brachiata and further identified as Bacillus subtilis 4NP-BL by phylogenetic analysis of 16S rRNA gene sequences. The maximum melanin yield of up to 1.5 g dry wt L-1 of production media was obtained through central composite design (CCD). The isolated melanin belonged to the eumelanin class with an irregular structure on the basis of elemental analysis, UV-vis, Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), and NMR studies. Furthermore, purified melanin displayed antioxidant activity and antimicrobial activity against pathogens Xanthomonas campestris and Alteromonas macleodii. Thus, this study further suggests a probable role of endophytes that produce melanin in aiding host plant protection from environmental stress and other pathogens.